This is a competing renewal application focused on the mechanisms of leukemogenesis and the identification of new targets for the treatment of acute myeloid leukemia (AML). Developing novel therapeutic approaches for the treatment of AML is of high clinical translational relevance and importance, as the outcome for the majority of patients with acute myeloid leukemia (AML) remains very poor, despite recent advances in the field. The emergence of leukemic cell resistance continues to be a serious problem and identifying pathways that can be targeted to eliminate leukemia stem cells (LSCs) would be of high relevance and importance. Work from our laboratory has provided evidence for the existence of negative feedback regulatory loops in myeloid leukemia cells that are engaged in response to chemotherapy or other antineoplastic agents and mediate leukemic cell resistance. These include activation of mitogen activated protein kinase (MAPK) cascades and other regulatory negative feedback loops. The kinases MNK1 and MNK2 are key effectors of MAPK pathways and control phosphorylation of the eukaryotic initiation factor 4E (eIF4E), a key element of the cap-translation initiation complex, whose function is critical for malignant transformation and survival of neoplastic cells. In addition, work from our group has provided the first evidence that MNK kinases are activated in a negative feedback regulatory manner to mediate eIF4E phosphorylation and to promote survival of primitive leukemic precursor cells in AML. Because of such key roles for eIF4E in tumorigenesis, targeting this signaling cascade using MNK kinase inhibitors may provide a unique approach to target and eliminate LSCs. The current proposal is a systematic approach to define the mechanisms by which MNK kinases promote LSC survival in AML and aims to use such information towards identifying novel cellular elements to selectively target LSCs and develop new therapeutic approaches for AML. Specific aim 1 will define MNK effector pathways in AML leukemic progenitors and will dissect their contributions in leukemogenesis. Experiments will be performed to define the roles of MNK-regulated effectors in controlling oncogenic mRNA translation, processing, cell proliferation, and survival of leukemic precursors. In addition the differential requirement of MNK1 versus MNK2 in leukemogenesis and their regulatory effects on downstream pathways will be dissected. Specific Aim 2 will examine the roles of MNK kinases and effector pathways in antileukemic responses in AML models in vivo. AML mouse models will be established in single Mnk1-/- or Mnk2-/- or double Mnk1-/-2-/- knockout mice, and the impact of different MNK kinases in leukemogenesis and generation of antileukemic responses in response to chemotherapy and other antileukemic agents will be addressed. Similar studies will be performed using mutant eIF4E knock-in mice, in which eIF4E cannot undergo MNK-mediated phosphorylation. Specific aim 3 examine the antileukemic properties of novel MNK inhibitors on primary leukemic precursors and LSCs from AML patients and will attempt to target negative feedback loops to enhance responses. The effects of novel MNK inhibitors on primary cells from a large number of patients with AML will be examined and their effects on survival of LSCs will be defined. The activation of negative feedback pathways will be also assessed and the effects of combinations of MNK inhibitors with mTOR targeting agents or other modulators of such feedback loops on leukemic stem cell survival will be defined. Altogether, these studies should advance our understanding of the mechanisms of leukemogenesis and provide the basis for important future clinical-translational efforts involving the use of novel inhibitors targeting MNK kinases or their effectors for the treatment of AML.